Techniques are now well established for directing E. coli-produced heterologous proteins across the inner membrane of the E. coli cell envelope, into a space between the inner and the outer membrane known as the periplasm. It has been shown that when a protein is expressed as a fusion protein, having an E. coli-recognized peptide or "signal peptide" attached to its N-terminus, the desired protein is secreted into the periplasm (see EP 177,343 Genentech Inc.). Several such signal peptides have now been identified by amino acid and DNA sequence (see Watson, M. Nucleic Acids Research, Vol 12, No. 13, 1984, pp. 5145-5164) and have been used successfully to direct heterologous proteins into the E. coli periplasm (see Oka et al, Proc. Natl. Acad. Sci. U.S.A., Vol 82, pp 7212-7216, November 1985 in which human epidermal growth factor (hEGF) was fused with the signal peptide of E. coli alkaline phosphatase to direct mature EGF into the E. coli periplasm; and see Hsiung et al, Biotechnology, Vol 4, November 1986, pp 991-995 where human growth hormone was directed into the E. coli periplasm using the signal peptide of outer membrane protein A (ompA) of E. coli).
While these periplasmic proteins are often described as "secreted" it should be understood that they are contained intracellularly by the outer membrane and, because they are not available in the medium, can be recovered only once the outer membrane is either disrupted or "permeabilized" to release the periplasmic components.
To facilitate the protein recovery process it is preferable that the protein accumulates in the culture medium during culturing of the protein-producing E. coli host, since there are relatively fewer contaminants and the protein can be recovered without damaging its cellular source. There have been various attempts at developing such a system, for excreting proteins to the medium, all of which include some tactic for overcoming the integrity of the outer membrane barrier.
One approach generally involves genetically engineering E. coli to co-express the desired protein in secretable form (bearing a signal peptide) together with a lytic protein which affects outer membrane integrity to the extent that it becomes permeable or "leaky" (see U.S. Pat. No. 4,595,658 in which the gene III product of the fI phage is used as the permeabilizing agent; Kobayashi et al, J. Bacteriol. June 1986, pp 728-732 in which activation of the cryptic kil protein is used; and see EP 140,864 in which a temperature sensitive lysogen is used). In these methods, careful control of lytic product expression is required to maintain host viability. Moreover, co-expression per se can be an energy drain on the host, reducing yield of the desired protein and reducing host cell biomass.
In another approach, the desired protein is expressed as a chimeric protein, in which the desired protein is fused to a carrier protein. The carrier proteins typically used are those naturally produced E. coli proteins, or fragments thereof, which are capable of being excreted (hemolysin) or which are bound to the outer membrane (ompF). For example, Mackman et al found that ompF, without its signal peptide, could be dragged through the E. coli envelope to the medium when fused with a C-terminal portion of hemolysin (EMBO J. Vol 6, No. 9, pp. 2835-2841, 1987). Nagahari et al found that human .beta.-endorphin was released to the medium when fused with at least a portion of the E. coli ompF protein (EMBO J. Vol 4, No. 13A, pp. 3589-3592, 1985). To recover proteins produced by such techniques, however, the desired protein must be cleaved from the carrier protein, which makes isolation difficult.
Yet another approach makes use of E. coli strains in which the integrity of the outer membrane has been compromised at the genetic level. Such strains, known as "leaky" hosts, are incapable of retaining periplasmic proteins. In practise, these strains are difficult to maintain in a viable condition without stringent control of environmental conditions.
It is an object of the present invention to provide a method enabling heterologous proteins to be excreted to the culture medium in which the protein-producing E. coli host is grown.
It is an object of the present invention to provide a DNA vector enabling excretion from E. coli of a heterologous protein encoded thereon.
It is a further object of the invention to provide an E. coli strain that has been genetically engineered to express a desired heterologous protein which is excreted to the medium in which the E. coli strain is cultured.
It is an overall object of the present invention to facilitate recovery of E. coli-produced, heterologous proteins by enabling accumulation of those proteins in the E. coli culture medium.